Derivatisation of granulocyte colony-stimulating factor

ABSTRACT

The present invention relates to a compound which is a polysaccharide derivative of GCSF, or of a GCSF like protein, wherein the polysaccharide is anionic and comprises between 2 and 200 saccharide units. The present invention also relates to pharmaceutical compositions comprising the novel compounds, and methods for making the novel compounds.

The present invention relates to novel polysaccharide derivatives ofGCSF and methods for producing such derivatives. The derivatives areuseful for improving the stability, pharmacokinetics andpharmacodynamics of GCSF.

Granulocyte Colony-Stimulating Factor (GCSF, CSF3) is a glycoprotein. Itmay act as a hormone, growth factor or cytokine and is produced by anumber of different tissues to stimulate the bone marrow to producegranulocytes and stem cells. GCSF also stimulates the survival,proliferation, differentiation, and function of neutrophil precursorsand mature neutrophils.

GCSF is produced by endothelium, macrophages, and a number of otherimmune cells. The natural human glycoprotein exists in two forms, a 174-and 180-amino-acid-long protein of molecular weight 19,800 grams permole. The more-abundant and more-active 174-amino acid form has beenused in the development of pharmaceutical products by recombinant DNA(rDNA) technology. Mouse GCSF was first recognised and purified inAustralia in 1983, and the human form was cloned by groups from Japanand the United States in 1986. The GCSF-receptor is present on precursorcells in the bone marrow, and in response to stimulation by GCSF,initiates proliferation and differentiation into mature granulocytes.

GCSF stimulates the production of white blood cells. In oncology andhematology, a recombinant form of GCSF is used with certain cancerpatients to accelerate recovery from neutropenia after chemotherapy,allowing higher-intensity treatment regimens. Chemotherapy can causemyelosuppression and unacceptably low levels of white blood cells,making patients prone to infections and sepsis. GCSF is also used toincrease the number of hematopoietic stem cells in the blood beforecollection by leukopheresis for use in hematopoietic stem celltransplantation.

The recombinant human GCSF synthesised in an E. coli expression systemis called filgrastim. The structure of filgrastim differs slightly fromthe structure of the natural glycoprotein. Most published studies haveused filgrastim. Filgrastim (Neupogen®) is a commercially-available formof rhGCSF (recombinant human GCSF).

Another form of recombinant human GCSF, lenograstim, is synthesised inChinese Hamster Ovary cells. As this is a mammalian cell expressionsystem, lenograstim is indistinguishable from the 174-amino acid naturalhuman GCSF. No clinical or therapeutic consequences of the differencesbetween filgrastim and lenograstim have yet been identified, and therehave been no formal comparative studies.

Attempts have been made to derivatise GCSF to improve itspharmacokinetic properties. There is a product on the market,PEG-filgrastim (Neulasta®), which is a polyethyleneglycol derivatisedform of GCSF. This has been shown to have a longer half-life thanfilgrastim, reducing the necessity of daily injections. The design anddevelopment of PEG-filgrastim is described further in Curr. Pharm Des.2004; 10(11): 1235-44.

US20070014759 describes conjugates between GCSF and PEG moieties whichare linked via an intact glycosyl linking group. The conjugates areformed from both glycosylated and unglycosylated peptides by the actionof a glycosyltransferase on mid-chain amino acids. U.S. Pat. No.6,956,027 provides conditions for selectively modifying the N-terminusof GCSF with PEG.

Others have derivatised GCSF with molecules other than PEG. WO2005/014050, for instance, describes GCSF covalently linked tohydroxyalkyl starch.

In view of the prior art, there is a need to provide improvedderivatives of GCSF which can be used in human and animal therapy andhave optimised stability, half-lives and low toxicity. We have foundthat attaching PSAs to GCSF imparts such properties and have therebyarrived at this invention. This is the first time that GCSF linked atthe N-terminus to anionic polysaccharides has been described.

Polysialic acids (PSAs) are naturally occurring unbranched polymers ofsialic acid produced by certain bacterial strains and in mammals incertain cells. They can be produced in various degrees of polymerisationfrom n=about 80 or more sialic acid residues down to n=2 by limited acidhydrolysis or by digestion with neuraminidase, or by fractionation ofthe natural, bacterially derived forms of the polymer.

In recent years, the biological properties of polysialic acids,particularly those of the alpha-2,8 linked homopolymeric polysialicacid, have been exploited to modify the pharmacokinetic properties ofprotein and low molecular weight drug molecules. Polysialic acidderivatisation gives rise to dramatic improvements in half-life for anumber of circulating therapeutic proteins including catalase andasparaginase, and also allows such proteins to be used in the face ofpre-existing antibodies raised as an undesirable (and sometimesinevitable) consequence of prior exposure to the therapeutic protein[Fernandes and Gregoriadis, 2006; Jain et. al., 2003, 2004]. Thealpha-2,8 linked polysialic acid offers an attractive alternative toPEG, being an immunologically invisible biodegradable polymer which isnaturally part of the human body, and which degrades, via tissueneuraminidases, to sialic acid, a non-toxic saccharide.

We have previously described methods for the attachment ofpolysaccharides (in particular PSA) to therapeutic agents such asproteins [U.S. Pat. No. 5,846,951; WO-A-0187922]. Some of these methodsdepend upon chemical derivatisation of the ‘non-reducing’ end of thepolymer to create a protein-reactive aldehyde moiety which reacts atprimary amine groups. A non-reducing sialic acid terminal unit, since itcontains vicinal diols, can be readily (and selectively) oxidised withperiodate to yield a mono-aldehyde form, which is much more reactivetowards proteins, and which comprises a suitably reactive element forthe attachment of proteins via reductive amination and otherchemistries. The reaction is illustrated in FIGS. 1 and 2 wherein

FIG. 1 shows the oxidation of colominic acid (alpha-2,8 linkedpolysialic acid from E. coli) with sodium periodate to form aprotein-reactive aldehyde at the non-reducing end; and

FIG. 2 shows the selective reduction of the Schiff's base with sodiumcyanoborohydride to form a stable irreversible covalent bond with theprotein amino group.

Unintentional by-products may be generated during the conventionalconjugation reactions described above by reaction of the colominic acidwith side chains of amino acids, for instance. These may be sufficientto be troublesome in the manufacture of chemically defined conjugatesrequired by regulatory authorities for therapeutic use in man andanimals.

It is not straightforward to purify the intended reaction product (forinstance the monopolysialylated product) away from the variousunintended products, since the physicochemical characteristics of mostof the reaction products are similar. This means that techniques such asion-exchange chromatography and gel-permeation chromatography (whichseparate on the basis of charge and size respectively) produce poorpurification profiles. This problem can be overcome by reducing theproduct complexity in the conjugation reaction. We have developed a newmethod for conjugation of polysaccharides to proteins whereby the highreactivity of the N-terminal of the protein can be utilized and whichavoids the product complexity obtained using the established method(FIGS. 1 and 2) of reductive amination of proteins with periodateoxidised natural colominic acid.

In accordance with a first aspect of this invention we provide acompound which is an N-terminal polysaccharide derivative of GCSF, or ofa GCSF like protein, wherein the polysaccharide is anionic and comprisesbetween 2 and 200 saccharide units.

Hereinafter, when using the term GCSF, we also intend to cover GCSF-likeproteins. By GCSF-like protein, we mean any biological compoundpossessing the activity of human granulocyte colony stimulating factor(a) whose amino acid sequence is at least fifty percent (50%) identicalto SEQ I.D. No. 1, and (b) that has at least thirty-five percent (35%)preferably at least 50%, more preferably at least 60 or 70% humangranulocyte colony stimulating factor activity as measured by a bioassayin comparison to the World Health Organization International Standardfor human granulocyte colony stimulating factor (Human, rDNA-derived) asmeasured according to the bioassay described in Example 1.

GCSF-like proteins may also be referred to as “GCSF-homologues”. Whethertwo sequences are homologues is routinely calculated using a percentagesimilarity or identity, terms that are well known in the art. Sequencesshould be compared to SEQ ID NO. 1, which is human GCSF with Swissprotaccession number P09919. The active GCSF is residues 30-207 of thissequence. GCSF homologue sequences may either be compared to the wholeof SEQ ID No. 1, or to residues 30-207 thereof. Preferably, thehomologue is compared to the active GCSF.

In this invention homologues have 50% or greater similarity or identityat the amino acid level, more preferably 60%, 70%, 80% or greater, morepreferably 90% or greater, such as 95% or 99% identity or similarity atthe amino acid level. A number of programs are available to calculatesimilarity or identity; preferred programs are the BLASTn, BLASTp andBLASTx programs, run with default parameters, available atwww.ncbi.nlm.nih.gov. For Example, 2 amino acid sequences may becompared using the BLASTn program with default parameters (score=100,word length=11, expectation value=11, low complexity filtering=on). Theabove levels of homology may be calculated using these defaultparameters.

The GCSF may be glycosylated or non-glycosylated.

In this invention, the term GCSF includes natural GCSF extracted from ahuman or mammalian body and synthetic versions thereof, such asrecombinant human GCSF, for instance filgrastim and lenograstim asdiscussed above. Mutants of GCSF that have appropriate GCSF-likeactivity, such as cysteine mutants, are also included.

By “N-terminal derivative”, we mean that the GCSF is derivatised by theanionic polysaccharide at its N-terminal amine group.

Preferably, the polysaccharide has at least 2, more preferably at least5, most preferably at least 10, for instance at least 50 saccharideunits.

The anionic polysaccharide is preferably selected from polysialic acid,heparin, hyaluronic acid and chondroitin sulphate. Preferably, thepolysaccharide is polysialic acid and consists substantially only ofsialic acid units. However, the polysaccharide may have units other thansialic acid in the molecule. For instance, sialic acid units mayalternate with other saccharide units. Preferably, however, thepolysaccharide consists substantially of units of sialic acid.

Preferably the polysaccharide has a terminal sialic acid group, and asdetailed above, is more preferably a polysialic acid, that is apolysaccharide comprising at least 2 sialic acid units joined to oneanother through α-2-8 or α-2-9 linkages. A suitable polysialic acid hasa weight average molecular weight in the range 2 to 200 kDa, preferablyin the range 5 to 75 kDa. Most preferably, the polysialic acid isderived from a bacterial source, for instance polysaccharide B of E.coli KI, N. meningitidis, Maraxella liquefaciens or Pasteurellaaeruginosa or K92 polysaccharide from E. coli K92 strain. It is mostpreferably colominic acid from E. coli K1.

The polysialic acid may be in the form of a salt or the free acid. Itmay be in a hydrolysed form, such that the molecular weight has beenreduced following recovery from a bacterial source.

The polysaccharide, preferably polysialic acid, may be material having awide spread of molecular weights such as having a polydispersity of morethan 1.3, for instance as much as 2 or more. Preferably thepolydispersity of molecular weight is less than 1.3 or 1.2, morepreferably less than 1.1, for instance as low as 1.01.

Typically, the compound of this invention is a polysialic acidderivative of GCSF and comprises 80-180 sialic acid units. Moretypically, the compound comprises 100-150 sialic acid units. Preferably,the compound comprises 120-145, most preferably 130-140 sialic acidunits.

The compound according to the first aspect of this invention may be acovalently-linked conjugate between the N-terminus of GCSF or aGCSF-like protein and an anionic polysaccharide. Other means ofassociation between the polysaccharide and the GCSF includeelectrostatic attraction. However, covalent bonding is preferred. Thecovalent linkage may be an amide linkage between a carboxyl group and anamine group. Another linkage by which the GCSF could be covalentlybonded to the polysaccharide is via a Schiff base. Suitable groups forconjugating to amines are described further in WO 2006/016168.

The polysaccharide may be linked to the GCSF via either its reducing ornon-reducing terminal unit. One polysaccharide chain may be linked atboth terminal units to GCSF proteins. This means that one polysaccharidechain may be linked to two GCSF proteins, i.e. be derivatised at bothits reducing and non reducing end.

In the invention the polysaccharide may be a naturally occurringpolysaccharide, or a derivative of a naturally occurring polysaccharide,for instance, a polysaccharide which has been derivatised by a reactionof one or more active groups on the saccharide residues, or which hasbeen covalently linked to a derivatising group at the end of thepolysaccharide chain.

Methods for attaching polysaccharides to proteins are well known in theart and are described in more detail in WO 92/22331 and WO-A-0187922.The preferred methods in this invention are described in more detailbelow. Methods are also described in FIGS. 1 and 2 of this application.

The polysaccharide may be linked to the GCSF or GCSF-like proteindirectly, i.e. as shown in FIGS. 1 and 2, or via a linker. Suitablelinkers are derived from N-maleimide, vinylsulphone, N-iodoacetamide,orthopyridyl or N-hydroxysuccinimide-containing reagents. The linker mayalso be biostable or biodegradable and comprise, for instance, apolypeptide or a synthetic oligomer. The linker may be derived from abifunctional moiety, as further described in WO 2005/016973. A suitablebifunctional reagent is, for instance, Bis-NHS. The reagent may havegeneral formula Z—R¹—Z wherein each Z is a functional group and may bethe same or different and R¹ is a bifunctional organic radical.Preferably, R¹ is selected from the group consisting of alkanediyl,arylene, alkarylene, heteroarylene and alkylheteroarylene, any of whichmay substituted and/or interrupted by carbonyl, ester, sulfide, ether,amide and/or amine linkages. Particularly preferred is C₃-C₆ alkanediyl.Most preferably, R¹ corresponds to the appropriate portion of thesuitable bifunctional reagent

We provide in accordance with a second aspect of this invention acompound of general formula (I)

wherein m is at least one;

XB is derived from B−XH which is GCSF or a GCSF-like protein wherein XHis NH₂ or SH;

L is a bond, a linking group, or comprises a polypeptide or a syntheticoligomer;

GlyO is an anionic saccharide unit;

wherein the linking group, if present, is of general formula—Y—C(O)—R¹—C(O)—;

wherein Y is NR² or NR²—NR² and R¹ is a difunctional organic radical asdefined above; and R² is H or C₁₋₆ alkyl.

In this aspect of the invention the GCSF is linked to the non-reducingend of the polysaccharide. The terminal polysaccharide unit is a sialicacid unit. The other saccharide units in the polysaccharide arerepresented by GlyO and may be the same or different. Suitablesaccharide units include heparin, hyaluronic acid or chondroitinsulphate.

When the GCSF is attached directly to the polysaccharide, the group L isa bond. However, the group L may alternatively be derived from anN-maleimide, vinylsulphone, N-iodoacetamide, orthopyridyl orN-hydroxysuccinimide containing reagent. The reagent may have generalformula Z—R¹—Z as defined above. In this embodiment, L is typically agroup

Preferably, XH is NH₂ and is the N-terminal amine of the GCSF orGCSF-like protein. Alternatively, NH₂ may be the primary amine of alysine amino acid side chain. In a different embodiment, XH is a thiolgroup, SH, from the side chin of a cysteine amino acid.

Another aspect of the invention is a pharmaceutical compositioncomprising a novel compound as defined above and one or morepharmaceutically acceptable excipients.

The pharmaceutical composition may be in the form of an aqueoussuspension. Aqueous suspensions contain the novel compounds in admixturewith excipients suitable for the manufacture of aqueous suspensions. Thepharmaceutical compositions may be in the form of a sterile injectableaqueous or homogeneous suspension. This suspension may be formulatedaccording to the known art using suitable dispersing or wetting agentsand suspending agents.

Pharmaceutical compositions may be administered orally, intravenously,intraperitoneally, intramuscularly, subcutaneously, intranasally,intradermally, topically or intratracheally for human or veterinary use.

The compositions may further comprise a formulation additive. Byformulation additive we mean an excipient which is capable ofstabilising the GCSF either internally or externally, as described inWang et al (1999). The excipient may be a stabiliser, a solubilser or ametal ion. Suitable examples of formulation additives include one ormore buffers, stabilisers, surfactants, salts, polymers, metal ions,sugars, polyols or amino acids. These may be used alone or incombination.

Stabilisers typically act by destabilisation of the denatured state of aprotein leading to increased Gibbs free energy change for unfolding ofthe protein. The stabiliser is preferably a sugar or a polyol, forexample sucrose, sorbitol, trehalose, glycerol, mannitol, lactose andethylene glycol. A stabitling buffer is sodium phosphate.

The solubiliser is preferably a surfactant, preferably a non-Ionicsurfactant. Suitable examples include Tween 80, Tween 20, Tween 40,Pluoronic F68, Brij 35 and Triton X100.

The metal ion is preferably divalent. Suitable metal ions include Zn²⁺,Ni²⁺, Co²⁺, Sr²⁺, Cu²⁺, Ca²⁺, Mg²⁺ and Fe²⁺.

The formulation additive may also be a polymer selected from PSA, PEG orhydroxy-beta-cyclodextrin.

Suitable amino acids and amino acid derivatives for use as theformulation additive include histidine, glycine, other similar aminoacids and sodium aspartate.

Another aspect of this invention is a composition comprising apopulation of anionic polysaccharide derivatives of GCSF or a GCSF-likeprotein, wherein the derivatives comprise between 2 and 200 saccharideunits and wherein the population consists substantially only ofN-terminal derivatives of the protein.

By “population” we mean that there is more than one polysaccharidederivative in the composition. The derivatives may comprise the same ordifferent numbers of saccharide units. Preferably, the polydispersity ofthe polysaccharide in the composition is less than 1.3, more preferablyless than 1.1. Preferred polysaccharides are as detailed above for theother aspects of this invention.

In the population, substantially all of the GCSF is derivatised at theN-terminal amine only. By this, we mean that 85%, preferably at least90%, most preferably at least 95% of the protein in the population isderivatised with PSA at the N-terminal amine only.

The degree of derivatisation at the N-terminus can be measured usingtechniques well known in the art, such as peptide mapping and EdmanDegradation.

A further aspect of the invention is a compound as described above foruse in therapy.

In accordance with a final aspect of the invention, we provide a methodfor producing a polysaccharide derivative of GCSF or of a GCSF-likeprotein wherein an anionic polysaccharide comprising 2-200 saccharideunits is chemically reacted with the GCSF or GCSF-like protein.

It will be noted in this aspect of the invention, the polysaccharide mayreact at any group on the GCSF or GCSF-like protein. For instance, thepolysaccharide may react with an amine, amide, aryl, aldehyde, ketone,guanidino, midazole, hydroxyl, carboxyl or sulfhydryl group. Preferably,the group is an amine group, more preferably a terminal amine group. Theamine may alternatively be the amine side chain of an amino acid, suchas a lysine amino acid. The polysaccharide may also react at anycarbohydrate residues on the GCSF, such as on pendant glycon groups.

Polysaccharides may be linked to amino acid side chains by methods knowin the art. For instance, a polysaccharide may be coupled to theC-terminus, —COOH or carboxyl side chains of Asp or Glu by in vitrocoupling. Thiol groups of cysteine amino acids may also be linked topolysaccharides by in vitro coupling. These methods are describedfurther in WO03/055526, in particular the table on pages 6 and 7. Inthis reference, in vitro coupling is also used to link anoligosaccharide moiety to the amide group on the side chain of Gln. Invitro coupling methods for linking of oligosaccharide moieties toguanidino and imidazole groups of Arg and His residues respectively arealso described. Each of these methods may be used to derivatise the GCSFof the present invention.

The polysaccharide may also react with a modified form of GCSF. Forinstance, one or more groups on the GCSF may have undergone a chemicaltransformation, for instance, by reduction or oxidation. A reactivecarbonyl may be generated in the place of the terminal amino group ofGCSF using oxidation conditions, for instance.

Suitable polysaccharides for use in the method of this invention are asdescribed previously for the novel compounds.

The compounds of the invention may be manufactured by any of thesuitable methods described in the prior art. For example, a typicalmethod is described to our previous patent application WO 92/22331.

Typically, the anionic polysaccharide has been activated beforederivatisation to GCSF. It may, for instance, have a reactive aldehydegroup and the derivatisation reaction may be carried out under reducingconditions. The reactive aldehyde group may be produced by controlledoxidation of a hydroxyl group of the polysaccharide. Most preferablythis reactive aldehyde is generated in a preliminary step, in which thepolysaccharide is reacted under controlled oxidation conditions, forinstance using sodium periodate, in aqueous solution. Preferably theoxidation is a chemical oxidation, although enzymes which are capable ofcarrying out this step may also be used. The reactive aldehyde group maybe at the non-reducing end or reducing end of the polysaccharide. TheGCSF, typically the N-terminus, may then react with the reactivealdehyde group to produce an adduct which, when reduced, produces theN-terminal derivative of GCSF.

The activation of the polysaccharide should preferably be carried outunder conditions such that there is substantially no mid-chain cleavageof the backbone of the polysaccharide, that is substantially nomolecular weight reduction. The oxidant is suitably perruthenate, or,preferably, periodate. Oxidation may be carried out with periodate at aconcentration in the range 1 mM to 1M, at a pH in the range 3 to 10, atemperature in the range 0 to 60° C. for a time in the range 1 min to 48hours.

Suitable reduction conditions for the derivatisation reaction mayutilise hydrogen with catalysts or, preferably hydrides, such asborohydrides. These may be immobilised such as Amberlite (trademark)-supported borohydride. Preferably alkali metal hydrides such assodium borohydride is used as the reducing agent, at a concentration inthe range 1 μM to 0.1M, a pH in the range 5.0 to 10, a temperature inthe range 0 to 60° C. and a period in the range 1 min to 48 hours. Thereaction conditions are selected such that pendant carboxyl groups onthe starting material are not reduced. Other suitable reducing agentsare cyanoborohydride under acidic conditions, e.g. polymer supportedcyanoborohydride or alkali metal cyanoborohydride, L-ascorbic acid,sodium metabisulphite, L-selectride, triacetoxyborohydride etc.

Other activated derivatives of polysaccharides may have utility in thepresent invention, including those with pendant functional groups suchas NHS, as described in our earlier patent application WO 06/00540.

In one embodiment, the reactive aldehyde is at the reducing end of thepolysaccharide and the non-reducing end has been passivated such that itdoes not react with pendant groups on the GCSF.

The reactivity of the reducing end of colominic acid, though weaktowards protein targets, is sufficient to be troublesome in themanufacture of chemically defined conjugates.

Chemistry suitable for preparing a polysaccharide with a reactivealdehyde at the reducing terminal of a polysaccharide is described inour earlier application WO 05/016974. The process involves a preliminaryselective oxidation step followed by reduction and then furtheroxidation to produce a compound with an aldehyde at the reducingterminal and a passivated non-reducing end.

WO 2005/016973 describes polysialic acid derivatives that are useful forconjugation to proteins, particularly those which have free sulfhydryldrugs. The polysialic acid compound is reacted with a heterobifunctionalreagent to introduce a pendant functional group for site-specificconjugation to sulfhydryl groups. The anionic polysaccharides used inthe present invention may also be derivatised with a heterobifunctionalreagent in this manner.

The polysaccharide may be derivatised before it reacts with GCSF. Forinstance, the polysaccharide may react with a bifunctional reagent.

The polysaccharide may be subjected to a preliminary reaction step, inwhich a group selected from a primary amine group, a secondary aminegroup and a hydrazine is formed on the terminal saccharide, which ispreferably sialic acid, followed by a reaction step in which this isreacted with a bifunctional reagent to form a reaction-intermediate, asfurther described in WO 2006/016168. The intermediate may then reactwith the GCSF or GCSF-like protein. The bifunctional reagent may havegeneral formula Z—R¹—Z, as defined previously.

We have found that certain reaction conditions promote selectivederivatisation at the N-terminal of the GCSF. To promote selectivereaction at the N-terminal, the derivatisation reaction should becarried out in a first aqueous solution of acidic pH, and the resultantpolysaccharide derivative should then be purified in a second aqueoussolution of higher pH than the first aqueous solution. Typically the pHof the first aqueous solution is less than 7, and is preferably in therange 4.0-6.0. The pH of the second aqueous solution is in the range of6.5-9.5, preferably 6.5-8.5. The low pH of the derivatisation reactionpromotes selective derivatisation at the N-terminus of the proteinrather than at any mid-chain sites.

Furthermore, we have found that the use of certain formulation additivespromotes the formation of a selective, stable, polysaccharideGCSF-derivative. The formulation additive may be selected from one ormore buffers, stabilisers, surfactants, salts, polymers, metal ions,sugars, polyols or amino acids. These may be added to the reactionmedium, or alternatively may be added to the final product composition,as a stabiliser.

In one embodiment of this invention, the formulation additive issorbitol, mannitol, trehalose or sucrose. In a different embodiment, theformulation additive is a non-ionic surfactant. The formulation additivemay alternatively be a polymer selected from PSA, PEG orhydroxy-beta-cyclodextrin e.g. Tween 20, Tween 80, PEG. In a differentembodiment the formulation additive is a divalent metal ion. Preferreddivalent metal ions include Zn²⁺, Ni²⁺, Co²⁺, Sr²⁺ or Fe²⁺.

The formulation additive may be a buffer. Preferably when theformulation additive is a buffer, it is sodium phosphate or sodiumacetate.

The purification of the polysaccharide derivative in the method of thepresent invention may be carried out using a variety of methods known inthe art. Examples of suitable purification methods include HIC(hydrophobic interaction chromotography), SEC (size exclusionchromotography), HPLC (high performance liquid chromotography), AEX(anion exchange chromotography) and MAC (metal affinity chromatography).

A population of polysialic acids having a wide molecular weightdistribution may be fractionated into fractions with lowerpolydispersities, i.e. into fractions with differing average molecularweights. Fractionation is preferably performed by anion exchangechromatography, using for elution a suitable basic buffer, as describedin our earlier patent applications WO 2005/016794 and WO 2005/03149. Thefractionation method is suitable for a polysaccharide starting materialas well as to the derivatives. The technique may thus be applied beforeor after the essential process steps of this invention. Preferably, thepolydispersity of the resultant polysaccharide derivative of GCSF isless than 1,1.

The derivatisation of GCSF in accordance with this invention, results inincreased half life, improved stability, reduced immunogenicity, and/orcontrol of solubility and hence bioavailability and the pharmacokineticproperties of GCSF. The new method is of particular value for creationof a monopolysialylated-GCSF conjugates.

The invention is illustrated by Examples 1-10 and by reference to thefollowing drawings:—

FIG. 1 is a reaction scheme showing the prior art activation of thenon-reducing sialic acid terminal unit;

FIG. 2 is a reaction scheme showing the N-terminal or randomderivatization of proteins;

FIG. 3 a shows the degradation of 24 kDa colominic acid (CA) atdifferent pHs using Triple Detection GPC (Viscotek:RI+RALS+Viscosometer);

FIG. 3 b shows the results of GPC chromatography of CA fractionated with400 mM NaCl;

FIG. 4 shows the characterisation of polysialylated GCSF by SDS-PAGE inthe presence of formulation additives;

FIG. 5 shows the characterisation of SE-HPLC (left hand side) andpolysialylated GCSF by SDS-PAGE (right hand side);

FIG. 6 shows the characterisation of GCSF by SE-HPLC;

FIG. 7 shows the characterisation of polysialylated 42 kDa-GCSF bynative PAGE;

FIG. 8 illustrates the results from immobilised metal affinitychromatography of GCSF;

FIG. 9 illustrates the in vitro activity of polysialylated GCSF onMNFS60 cells;

FIG. 10 shows a SE-HPLC data for the stability of polysialylated GCSFformulation (CA41 KDa-GCSF);

FIG. 11 shows the FACS data for GCSF (neutrophil count);

FIG. 12 shows the in vivo efficacy of GCSF formulations;

FIG. 13 shows the characterisation of pegylated GCSF by SE-HPLC.

EXAMPLES Material

Ammonium carbonate, ethylene glycol, polyethylene glycol (8 KDa), sodiumcyanoborohydride (>98% pure), sodium meta-periodate and molecular weightmarkers, ammonium sulphate, sodium chloride, sodium phosphate, sorbitol,Tween 20 and Tris were obtained from Sigma Chemical Laboratory, UK.Sodium acetate and sodium phosphate were from BDH, UK. The colominicacid used, linear alpha-(2,8)-linked E. coli K1 polysialic acids (22.7kDa average, high polydispersity 1.34, 39 kDa p.d. 1.4; 11 kDa, p.d.1.27) was from Camida, Ireland. Other materials included 2,4dinitrophenyl hydrazine (Aldrich Chemical Company, UK), dialysis tubing(3.5 KDa and 10 KDa cut off limits; Medicell International Limited, UK),Sepharose SP HiTrap, PD-10 columns, Q FF [column 1 ml or 5 ml]; HitrapButyl HP column [1 or 5 ml]; (Pharmacia, UK), Tris-glycinepolyacrylamide gels (4-20% and 16%). Tris-glycine sodium dodecylsulphaterunning buffer and loading buffer (Novex, UK). Deionised water wasobtained from an Elgastat Option 4 water purification unit (ElgaLimited, UK). All reagents used were of analytical grade. A plate reader(Dynex Technologies, UK) was used for spectrophotometric determinationsin protein or CA assays. Mice were purchased from Harlan, UK andacclimatized for at least one week prior to their use. GCSF was obtainedfrom SIIL, India.

Example 1 GCSF Bioassay

G-CSF biological activity determination is based on stimulation ofM-NFS-60 cells proliferation by this cytokine. Cells are incubated withserial dilutions of both reference and G-CSF preparations for 48 hours.Cells proliferative response is estimated after 4 hours incubation withviable cells dyeing system—PMS (electron coupling reagent) mixture. MTSis bioreduced by cells into formazan product that is soluble in tissueculture medium. The absorbance of formazan at 492 nm can be measureddirectly from 96 well assay plates without additional processing. Thequantity of formazan product, as measured by the amount of 492 nmabsorbance, is directly proportional to the number of living cells.

The World Health Organisation International Standard for GCSF (humanrDNA-derived) 88/502, 10,000 IU/ampoule, content 100 ng G-CSF, NIBSC, UKshould be used as the reference.

Example 2 Protein and Colominic Acid Determination

Quantitative estimation of polysialic acids (as sialic acid) with theresorcinol reagent was carried out by the resorcinol method[Svennerholm, 1957] as described elsewhere [Gregoriadis et al., 1993;Fernandes and Gregoriadis, 1996, 1997]. Protein was measured by the BCAcolorimetric method or UV absorbance at 280 nm.

21 Activation of Colominic Acid

Freshly prepared 0.02 M sodium metaperiodate (NalO₄) solution (8 foldmolar excess) was mixed with CA at 20° C. and the reaction mixture wasstirred magnetically for 15 min in the dark. A two-fold volume ofethylene glycol was then added to the reaction mixture to expend excessNaIO₄ and the mixture left to stir at 20° C. for a further 30 min. Theoxidised colominic acid was dialysed (3.5 KDa molecular weight cut offdialysis tubing) extensively (24 h) against a 0.01% ammonium carbonatebuffer (pH 7.4) at 4° C. Ultrafiltration (over molecular weight cut off3.5 kDa) was used to concentrate the CAO solution from the dialysistubing. Following concentration to required volume, the filterate waslyophilized and stored at −40° C. until further use. Alternatively, CAwas recovered from the reaction mixture by precipitation (twice) withethanol.

2.2 Determination of the Oxidation state of CA and Derivatives

Qualitative estimation of the degree of colominic acid oxidation wascarried out with 2,4 dinitrophenylhydrazine (2,4-DNPH), which yieldssparingly soluble 2,4 dinitrophenyl-hydrazones on interaction withcarbonyl compounds. Non-oxidised (CA)/oxidised (CAO) were added to the2,4-DNPH reagent (1.0 ml), the solutions were shaken and then allowed tostand at 37° C. until a crystalline precipitate was observed [Shrineret. al., 1980]. The degree (quantitative) of CA oxidation was measuredwith a method [Park and Johnson, 1949] based on the reduction offerricyanide ions in alkaline solution to ferric ferrocyanide (Persianblue), which is then measured at 630 nm. In this instance, glucose wasused as a standard.

2.3 Gel Permeation Chromatography

Colominic acid samples (CA and CAO) were dissolved in NaNO₃ (0.2M),CH₃CN (10%; 5 mg/ml) and were chromatographed on over 2×GMPW_(XL)columns with detection by refractive index (GPC system: VE1121 GPCsolvent pump, VE3580 RI detector and collation with Trisec 3 software(Viscotek Europe Ltd). Samples (5 mg/ml) were filtered over 0.45 μmnylon membrane and run at 0.7 cm/min with 0.2M NaNO₃ and CH₃CN (10%) asthe mobile phase.

The results are shown in FIG. 3 b and tables 4 and 5 (see page 25).

2.4 Colominic Acid Stability

The rules for chemistry of the PEGylation cannot be applied topolysialylation as such because of the difference in the physiochemicalproperties of these molecules. PSA is an acid labile polymer and isstable for weeks around neutral pH (FIG. 3 a). The results in FIG. 3 ashow that at pH 6.0 and 7.4 CA is stable for 8 days, at pH 5.0 there isslow degradation (after 48 hours 92% of initial MW), and at pH 4.0 thereis slow degradation (after 48 hours 70% of initial MW). Polysialic acidis highly hydrophilic whereas PEG is an ampiphilic molecule in nature.When the polysialylation is carried out using conditions used forPEGylation, aggregation and precipitation of the proteins is seen inmany cases.

Example 3 Preparation of N-terminal Protein-CA Conjugates withFormulation Additives 3.1 Preparation of GCSF-CA Conjugates (N-terminalMethod)

GCSF (18.8 kDa) was supplied as a solution (1.05 mg/ml in 10 mM sodiumacetate buffer, pH 4.0 containing 5% sorbitol, 0.025 mg/ml polysorbate80) and stored at 2-8° C. The required amount of GCSF was taken into aneppendorf and placed on ice. The amount of CA to be added forconjugation was calculated based on formula:

${{Weight}\mspace{14mu} {of}\mspace{14mu} {CA}} = {\frac{{Amount}\mspace{14mu} {of}\mspace{14mu} {{protein}(g)}}{( {{MW}\mspace{14mu} {of}\mspace{14mu} {protein}} )} \times ( {{MW}\mspace{14mu} {of}\mspace{14mu} {CA}} ) \times ( {{Molar}\mspace{14mu} {excess}\mspace{14mu} {of}\mspace{14mu} {CA}} )}$

Required amount of CA was weighed out. CA was solubilised in 10 mMNaOAc. 5% sorbitol, pH 5.5 (20% volume of the final reaction volume wasused here), gently vortexed the mixture until all the CA has dissolvedand then either filtered into a new eppendorf or centrifuged at 4000 rpmfor 5 min and the supernatant was transferred to a new eppendorf toremove any aggregated/precipitated material. Required volume of 10 mg/mlTween 20 stock solution was added, in order to have a finalconcentration of 0.5 mg/ml of the Tween 20 in the final reactionmixture. Required amount of GCSF protein solution was added to the CAsolution to give a 10 molar excess (small scale) and 9 (large scale) ofCA and gently mixed by keeping the reaction mixture on a gentle shakerat 4±1° C. 100 mg/ml NaCNBH₃ solution was added In order to have 50 mMor 3.17 mg/ml In the final reaction mixture, gently mixed and pH of thefinal reaction mixture was checked, if necessary adjusted the pH to 5.5with 1 M NaOH/HCl at 4±1° C. Finally adjusted the volume of the reactionusing 10 mM NaOAc, 5% sorbitol, pH 5.5 to give a protein concentrationof 0.67 mg/ml in the reaction mixture. Tube was sealed and stirred atdesired temperature (4±1° C.) for 24 hours. The reaction was stopped byan appropriate method and samples were taken out for in vitro activityassay on MNFS 60 cell. SDS-PAGE (using 4-20% Tris glycine gel), SE-HPLC(superose 6 column) and checked the pH of reaction mixture. To eliminateany precipitate the reaction mixture was centrifuged at 13000 rpm for 5min before SE-HPLC analysis and purification, preferred buffer forSE-HPLC was 0.1 M Na phosphate (pH 6.9).

Optimisation

Reductive amination was performed with a range of molecular weights ofCA (29-52 kda) on GCSF for N-terminal and random derivatisation. Rangeof process variables were studied for conjugation reactions: CAO 10-20(small scale) and 8-15 (large scale) molar excess; reagent=50-100 mMNaCNBH₃; reaction buffer=10 mM NaOAc; pH 5.0-7.4, formulationadditives=Tween 20 6 KDa/Peg 8 KDa (10M excess)/Tween 20+PEG 6 KDa;temperature=4±1° C., time=16-24 hours etc.

Optimised reaction conditions were found to be as following: CAO=10(small scale) and 9 (large scale) molar excess, reagent-50 mM NaCNBH₃,Reaction buffer=10 mM NaOAc pH 5.5, additives=0.5 mg/ml, tween 20,temperature=4±1° C., time=22 hours.

3.2. Purification and Characterization of GCSF-CA Conjugates (N-terminalMethod)

The remaining reaction mixture sample was diluted with AEX buffer A (20mM sodium acetate, 50 mM sodium chloride pH 5.0) (1.5 ml reactionmixture+9 ml of buffer A), the pH was checked and adjusted if requiredto pH 5.0, and loaded on the AEX column previously equilibrated with AEXbuffer A. The loading fractions were collected and labelled. The columnwas washed with AEX buffer A (at least 5 column volume), fractions werecollected (each fraction 1.5 column volume) and labelled. The productwas eluted with AEX buffer B (50 mM sodium phosphate, 0.65 M sodiumchloride, pH 7.0), fractions were collected (each fraction 1 columnvolume; 6 column) and labelled. If two consecutive fractions had noprotein content (UV280 nm), the next step was carried out. The sampleswere kept on ice during purification. The protein concentration wasanalysed by UV (280 nm) (Abs of 1 mg/ml of GCSF was about 0.872). Thesamples were taken for SDS-PAGE and SE-HPLC. To remove free CA from themixture, HIC was used. The samples were concentrated, if required.

The AEX fractions containing conjugate were pooled and (NH₄)₂SO₄ addedto give a concentration of 2.75 M in the loading solution. This solutionwas then loaded on to the HIC column previously equilibrated with HICbuffer A (10 mM sodium phosphate, 2.75 M ammonium sulphate, pH 6.5). Theloading fractions were collected (each fraction 1.5 column volume) andlabelled. The column was washed with HIC buffer A (at least 5 columnvolumes; rate=0.5 ml/min; (1.5 column volume) fractions collected andlabelled. The product was eluted with HIC buffer B (20 mM sodiumphosphate pH 7.4) (rate=5 ml/min); fractions were collected (1 columnvolume fraction; 6 column volume) and labelled. Samples were kept on iceduring purification. Protein concentration was analyzed by UV (280 nm).The HIC fractions containing the purified conjugate were combined andcomposition of the conjugate in solution was adjusted with 50% sorbitolsolution and 10 mg/ml Tween 20 solution to give a final composition of5% sorbitol and 0.025 mg/ml Tween 20. The solution was then concentratedat 4±1° C. and the protein concentration analysed by UV (280 nm).Further purification was done by SE-HPLC (e.g. to separate conjugatesfrom free protein/aggregates etc.). Conjugate were sterile filtered andsamples taken for activity assay and for characterisation by SDS-PAGEand SE-HPLC. If required an aliquot was removed for a protein assay andCA assay. The remainder was stored at 4±1° C. until further use andstudied for physical stability by SE-HPLC.

The effects of various processes affecting the stability of GCSF insolution and the degree of derivatization were studied.

3.3.1. Preparation of GCSF-CA Conjugates (Random)

GCSF (18.8 kDa) was supplied as a solution (1.05 mg/ml in 10 mM sodiumacetate buffer, pH 4.0 containing 5% sorbitol, 0.025 mg/ml polysorbate80) and stored at 2-8° C. The required amount of GCSF was taken into aneppendorf and placed on ice. The amount of CA (e.g. oxidised ornon-oxidised CA) to be added for conjugation was calculated based onformula:

${{Weight}\mspace{14mu} {of}\mspace{14mu} {CA}} = {\frac{{Amount}\mspace{14mu} {of}\mspace{14mu} {{protein}(g)}}{{MW}\mspace{14mu} {of}\mspace{14mu} {protein}} \times ( {{MW}\mspace{14mu} {of}\mspace{14mu} {CA}} ) \times ( {{Molar}\mspace{14mu} {excess}\mspace{14mu} {of}\mspace{14mu} {CA}} )}$

The required amount of CA was weighed out. The CA was solubilised in 50mM sodium phosphate, 5% sorbitol, pH 7.4 (20% volume of the finalreaction volume was used here). The mixture was gently vortexed untilall the CA has dissolved and then either filtered into a new eppendorfor centrifuged at 4000 rpm for 5 min and the supernatant transferred toa new eppendorf to remove any aggregated/precipitated material. Therequired volume of 10 mg/ml Tween 20 stock solution was added, in orderto have a final concentration of 0.5 mg/ml of the Tween 20 in the finalreaction mixture. The required amount of GCSF protein solution was addedto the CA solution to give a 11 molar excess (for 40 kDa) of CA andgently mixed by keeping the reaction mixture on a gentle shaker at 4±1°C. 100 mg/ml NaCNBH₃ solution was added in order to have 50 mM or 3.17mg/ml in the final reaction mixture, gently mixed and pH of the finalreaction mixture was checked, if necessary adjusted the pH to 7.4 with 1M NaOH/HCl at 4±1° C. Finally adjusted the volume of the reaction using10 mM NaOAc, 5% sorbitol, pH 7.4 to give a protein concentration of 0.67mg/ml in the reaction mixture. Tube was sealed and stirred at desiredtemperature (4±1° C.) for 22 hours. The reaction was stopped by anappropriate method and samples were taken out for in vitro activityassay on MNFS 60 cells, SDS-PAGE (using 4-20% Tris glycine gel), SE-HPLCand the pH of reaction mixture was checked. To eliminate any precipitatethe reaction mixture was centrifuged at 13000 rpm for 5 min beforeSE-HPLC analysis and purification, preferred buffer for SE-HPLC was 0.1M Na phosphate (pH 6.9).

3.32. Purification and Characterisation of GCSF-CA Conjugates (Random)

Monosialylated GCSF conjugates were purified from the other GCSFconjugates by HIC and IEC. The remaining reaction mixture sample wasdiluted with AEX buffer A (20 mM sodium acetate, 50 mM sodium chloridepH 5.0) (1.5 ml reaction mixture+9 ml of buffer A), the pH was checkedand adjusted if required to pH 5.0 and loaded on the AEX columnpreviously equilibrated with AEX buffer A. The loading fractions werecollected and labelled. The column was washed with AEX buffer A (atleast 5 column volume), fractions collected (each fraction 1.5 columnvolume) and labelled. The product was eluted with AEX buffer B (50 mMsodium phosphate, 0.65 M sodium chloride, pH 7.0), fractions collected(each fraction 1 column volume; 6 column) and labelled. If twoconsecutive fractions had no protein content (UV280 nm), the next stepwas followed. Samples were kept on ice during purification. The proteinconcentration was analysed by UV (280 nm) (Abs of 1 mg/ml of GCSF wasabout 0.872). The samples were taken for SDS-PAGE and SE-HPLC. To removefree CA from the mixture, HIC was used. Samples were concentrated, ifrequired.

The AEX fractions containing conjugate were pooled and (NH₄)₂SO₄ wasadded to give a concentration of 2.75 M In the loading solution. Thissolution was then loaded on to the HIC column previously equilibratedwith HIC buffer A (10 mM Sodium Phosphate, 2.75 M ammonium sulphate, pH6.5). The loading fractions were collected (each fraction 1.5 columnvolume) and labelled. The column was washed with HIC buffer A (at least5 column volumes; rate=0.5 ml/min. (1.5 column volume) fractions werecollected and labelled. The product was eluted with HIC buffer B (20 mMsodium phosphate pH 7.4) (rate=5 ml/min); fractions collected (1 columnvolume fraction; 6 column volume) and labelled. Samples were kept on iceduring purification. The protein concentration was analyzed by UV (280nm). The HIC fractions containing the purified conjugate were combinedand the composition of the conjugate in solution was adjusted with 50%sorbitol solution and 10 mg/ml Tween 20 solution to give a finalcomposition of 5% sorbitol and 0.025 mg/ml Tween 20. The solution wasthen concentrated at 4±1° C. and the protein concentration was analysedby UV (280 nm). Further purification was done by SE-HPLC (e.g. toseparate conjugates from free protein/aggregates etc.). The conjugateswere sterile filtered and samples taken for activity assay and forcharacterisation by SDS-PAGE and SE-HPLC. An aliquot was removed forprotein assay and CA assay. The remainder was stored at 4±1° C. untilfurther use and studied for physical stability by SE-HPLC.

The effects of various processes affecting the stability of GCSF insolution and the degree of derivatization were studied.

3.4. Pegylation of GCSF (Comparative):

GCSF (18.8 kDa) was supplied as a solution (0.5 mg/ml in 10 mM sodiumacetate buffer, pH 4.0 containing 5% sorbitol, 0.025 mg/ml polysorbate80) and stored at 2.8° C. GCSF solution was concentrated to make about1.0 mg/ml of solution. The required amount of GCSF was taken into aneppendorf and placed on ice. The amount of PEG added for conjugation wascalculated based on formula:

${{Weight}\mspace{14mu} {of}\mspace{14mu} {PEG}} = {\frac{{Amount}\mspace{14mu} {of}\mspace{14mu} {{protein}(g)}}{( {{MW}\mspace{14mu} {of}\mspace{14mu} {protein}} )} \times ( {{MW}\mspace{14mu} {of}\mspace{14mu} {PEG}} ) \times ( {{Molar}\mspace{14mu} {excess}\mspace{14mu} {of}\mspace{14mu} {PEG}} )}$

The required amount of PEG 20K was weighed out. It was solubilzed in 10mM NaOAc. 5% sorbitol, pH 5.5 (20% volume of the final reaction volumeas used here), the mixture gently vortexed until all the PEG haddissolved and then either filtered into a new eppendorf or centrifugedat 4000 rpm for 5 min and the supernatant was transferred to a neweppendorf to remove any aggregated/precipitated material. The requiredvolume of 10 mg/ml Tween 20 stock solution was added, in order to have afinal concentration of 0.5 mg/ml of the Tween 20 in the final reactionmixture. Required amount of GCSF protein solution was added to the PEGsolution to give a 7.5 molar excess of PEG and gently mixed by keepingthe reaction mixture on a gentle shaker at 4±1° C. 100 mg/ml NaCNBH₃solution was added in order to have 50 mM or 3.17 mg/ml in the finalreaction mixture, gently mixed and the pH of the final reaction mixturewas checked, and if necessary adjusted to 5.5 with 1M NaOH/HCL at 4±1°C. Finally adjusted the volume of the reaction using 10 mM NaOAC, 5%sorbitol, and pH 5.5 to give a protein concentration of 1 mg/ml in thereaction mixture. The tube was sealed and stirred at desired temperature(4±1° C.) for 24 hours. The reaction was stopped by an appropriatemethod and samples were taken out for in vitro activity on MNSF 60 cell,SDS-PAGE (using 4-20% Tris-glycine gel), SE-HPLC (superose 6 column) andchecked the pH of reaction mixture. To eliminate any precipitate thereaction mixture was centrifuged at 13000 rpm for 5 min before SE-HPLCanalysis and purification, preferred buffer for SE-HPLC was 0.1 M sodiumphosphate (pH 6.9). The results are shown in FIG. 13.

3.5. Metal Affinity Chromatography

GCSF and PSA-GCSF conjugates were purified from the PSA and by-productsof the reaction mixture by metal affinity chromatography (FIG. 8). Thesample for this experiment was 50 ug GCSF in 75 uL reaction buffer+75 uleluent A. The reaction buffer was 0.5 mg/mL Tween 20; 5% sorbitol; 10 mMNaOAc; pH 5.0. Eluent A was 10 mM Tris/HCl; pH 7.0. Eluent B was 20 mMAcOH+0.2 M NaCl; pH 4.0. Gradient: (t/min)=0 to 12.5 (100% A); t=12.5 to25 (30% B); 25 to 40 (100% B). The peak at 0.887 is the buffer, and thatat 16.142 is GCSF.

3.6. SE-HPLC of GCSF Formulations

HPLC was performed on a Liquid Chromatograph (JASCO) equipped with aJasco. AS-2057 plus autosampler refrigerated at 4° C., and a JascoUV-975 UV/VIS detector. Data was recorded by EZchrom Elite software onan IBM/PC. The SEC samples were analysed with an isocratic mobile phaseof 0.1 M Na phosphate, pH 6.9; on a Superose 6 column (FIG. 5) in thepresence of Tween. FIG. 6 shows just one peak at RT=76.408, which isattributed to GCSF.

The peak table for the SEC shown on the right hand side of FIG. 5 is asfollows:

TABLE 1 Peak RT % Area Species 1 33.896 13.9 Aggregate 2 60.871 85.7CA38K-GCSF 3 76.229 0.4 GCSF

3.7. Native, SDS Polyacrylamide Gel Electrophoresis & Western Blotting

SDS-PAGE was performed using 4-20% trisglycine gels. Samples werediluted with either reducing or non reducing buffer and 5.0 ug ofprotein was loaded into each well. The gels were run on a trisglycinebuffer system and was stained with Coomassie Blue. Western blotting wasperformed using anti PSA antibody (FIG. 4). FIG. 4 shows the SDS-PAGE ofGCSF formulations (site-specific; N-terminal). Native PAGE was performedon 10% tris glycine gel (FIG. 7).

3.8 In Vitro Activity

In vitro studies were performed on MNFS 60 cells with GCSF, PSA and PEGconjugates. EC50 values were measured and compared for various GCSFformulations (FIG. 9).

3.9. Stability Studies

Sterile GCSF conjugates were stored in 20 mM sodium phosphate, pH 7.4;5% sorbitol and 0.025 mg/ml Tween 20; at 4° C. for six weeks. SE-HPLC ofthe samples was performed every week using SEC columns under followingconditions: Injection volume 100 ul, flow rate 0.250 ml/min, runningbuffer 0.1 M sodium phosphate, pH 6.9 (FIG. 10).

3.10. In Vivo Efficacy of GCSF Formulations

The in vivo efficacy of GCSF formulations was studied in female miceB6D2F1, 7-8 weeks old, 5-15 ugs of protein dose (same activity) wasinjected in mice subcutaneously. Animals were divided into seven groupsof four. GCSF formulations were given to each animal of each group inthe following manner; GCSF (5 μg), GCSF (15 μg), GCSF-PSA conjugates(5-15 μg), PBS, GCSF-PEG20 (NeulastaR; 5 μg). 50 μl of blood was takenfrom each animal and was analysed by FACS after staining with antibodiesspecific for WBCs (FIGS. 11 and 12).

Results Activation of CA and Determination of Degree of Oxidation

Colominic acid (CA) is a linear alpha-2,8-inked homopolymer ofN-acetylneuraminic acid (Neu5Ac) residues was used. Exposure ofcolominic acids to oxidation was carried out for 15 min using 20 mMperiodate at room temperature. The integrity of the internal alpha-2,8linked Neu5Ac residues post periodate treatment was analysed by gelpermeation chromatography and the chromatographs obtained for theoxidised (CAO), material was compared with that of native CA. It wasfound that oxidized and native CA exhibit almost identical elutionprofiles, with no evidence that the successive oxidation step give riseto significant fragmentation of the polymer chain.

Quantitative measurement of the oxidation state of CA was performed byferricyanide ion reduction in alkaline solution to ferrocyanide(Prussian Blue) [Park and Johnson, 1949] using glucose as a standard.Table 2 shows that the oxidized colominic acid was found to have agreater than stoichiometric (>100%) amount of reducing agent, i.e. 112mol % of apparent aldehyde content comprising the combined reducingpower of the reducing end hemiketal and the introduced aldehyde (at theother end, reducing end).

TABLE 2 Degree of oxidation of various colominic acid intermediates inthe double oxidation reaction scheme using glucose as a standard (100%,1 mole of aldehyde per mole of glucose; n = 3 ± s.d). CA species Degreeof oxidation colominic acid (CA) 16.1 ± 0.63 colominic acid-oxidised(CAO) 112.03 ± 4.97  colominic acid-reduced (CAOR) 0; Not detectablecolominic acid-oxidised-reduced-oxidised (CAORO) 95.47 ± 7.11 

Preparation, Purification and Characterization of GCSF Conjugate

The procedure to prepare and purify colominic acid (CA) conjugates ofgranulocyte-colony stimulating factor (GCSF) in an N-terminallyselective manner by conducting the reaction at a reduced pH (pH 5.5) andat 4±1° C. is detailed above. This involves conjugation in the presenceof sodium cyanoborohydride, followed by purification using ion-exchangechromatography (AEX) to remove free GCSF followed by removal of CA byhydrophobic interaction chromatography (HIC). The low pH was used tofavour selective derivatisation of the alpha amino group of theN-terminus, and also in order to minimise aggregation of GCSF during thereaction. The composition of the final reaction buffer was 5% sorbitol,0.5 mg/ml Tween 20 in 10 mM NaOAc at pH 5.5.

Formation of the GCSF-CA conjugates and stability was confirmed by theSE-HPLC (change of retention time of GCSF-PSA as compared to GCSF; alsoco-elution of both moieties); ion exchange chromatography (binding ofconjugates on to the AEC column) and polyacrylamide gel electrophoresis(SDS-PAGE; shifting of bands with high m.w. species and native page).The conjugates used in the in vitro cell line assay (on MNFS-60 cells)were −40% active as compared native protein. The conjugates preparedwithout formulation additives led to the aggregation of protein withpoor degree of derivatization. FIG. 5, shows the SE-HPLC data forGCSF-CA 39 kDa reaction mixture after 24 hours, prepared in the presenceof Tween 20. The characterisation conditions were column: Superdex 200,buffer ammonium bicarbonate 0.15 M pH 7.8. The formation of GCSF-PEGconjugate was confirmed by SE-HPLC (FIG. 13). GCSF and PSA-GCSFconjugates were successfully purified from the PSA and by-products ofthe reaction mixture by metal affinity chromatography (FIG. 8). GCSFconjugates were found to be stable even after six weeks of storage in 20mM sodium phosphate, pH 7.4 (FIG. 10).

Table 3 shows the peak analysis of FIG. 5.

TABLE 3 Peak RT % Area Species 1 31.683 8.80 aggregate 2 42.683 11.77(CA)2-GCSF 3 49.058 68.55 CA-GCSF 4 68.833 10.89 GCSFTable 4 shows values of various parameters used and table 5 gives themolecular weight and polydispersity of CA fractions.

TABLE 4 Parameters Values Mn (Da) 26,666 Mw (Da) 27,956 Mz (Da) 31,129Mp (Da) 22,969 Mw/Mn 1.048 IV (dl/g) 0.2395 Rh (nm) 4.683 Branches 0.00Sample Conc (mg/ml) 5.600 Sample Recovery (%) 90.71 dn/dc (ml/g) 0.156dA/dc (ml/g) 0.000 Mark-Houwink a −0.048 Mark-Houwink logK −0.425

TABLE 5 CA fraction Mw (kDa) pd 475 97.2 1.285 450 52.3 1.109 425 37.91.062 400 28.0 1.048 375 19.0 1.080 *350 14.5 — *300 10.0 — *250 7.0 —

The PSA conjugates were found to be active in the in vitro activityassay (FIG. 9). In vivo efficacy study shows that PSA-GCSF conjugatesare as good as PEG conjugates and vastly superior to GCSF (FIGS. 11&12).

REFERENCES

-   Fernandes, A. I., Gregoriadis, G., Synthesis, characterization and    properties of polysialylated catalase, Biochimica at Biophysica    Acta, 1293 (1996) 92-96.-   Fernandes. A., Gregoriadis, G., Polysialylated asparaginase:    preparation, activity and pharmacokinetics, Biochimica at Biophysica    Acta, 1341 (1997) 26-34.-   Gregoriadis, G., McCormack, B., Wang, Z., Lifely, R., Polysialic    acids: potential in drug delivery, FEBS Letters, 315 (1993) 271-276.-   Jain et. al., Polysialylated insulin: synthesis, characterization    and biological activity in vivo. Biochemica et. Biophysica Acta,    1622 (2003) 42-49.-   Jain et. al., The natural way to improve the stability and    pharmacokinetics of protein and peptide drugs. Drug delivery systems    and sciences, 4(2), (2004) 3-9.-   Park, J. T., Johnson, M. J., A submicrodetermination of glucose,    Journal of Biological Chemistry, 181 (1949) 149-151.-   Shriner, R. L., Fuson, R. D. C., Curtin, D. Y., Morill, T. C., The    Systematic Identification of Organic Compounds, 6^(th) ed., Wiley,    New York, 1980.-   Svennerholm, L., Quantitative estimation of sialic acid II: A    colorimetric resorcinol-hydrochloric acid method, Biochemica at    Biophysica Acta, 24 (1957) 604.611.-   Wang, W., Instability, stabilization, and formulation of liquid    protein pharmaceuticals, International Journal of Pharmaceutics,    185 (1999) 129-188.

1. A compound which is an amino-terminal polysaccharide derivative ofgranulocyte colony-stimulating factor (GCSF), wherein the polysaccharideis attached to the amino terminus of the GCSF, wherein thepolysaccharide is anionic and comprises between 2 and 200 saccharideunits.
 2. The compound according to claim 1 wherein the polysaccharideis selected from polysialic acid, heparin, hyaluronic acid orchondroitin sulphate.
 3. The compound according to claim 1 wherein theGCSF is derivatised by the polysaccharide at the reducing terminal unitof the polysaccharide.
 4. A compound of formula (I)

wherein m is at least one; XB is B—XH wherein B is GCSF and XH is NH₂and is the N-terminal amine of the GCSF; L is a bond or a linking group;GlyO is an anionic saccharide unit; wherein the linking group, ifpresent, is of the formula—Y—C(O)—R¹—C(O)—; wherein R¹ is a difunctional organic radical selectedfrom the group consisting of alkanediyl, arylene, alkarylene,heteroarylene and alkylheteroarylene, any of which may substituted byand/or interrupted by carbonyl, ester, sulfide, ether, amide and/oramine linkages; and Y is NR² or NR²—NR² wherein R² is H or C₁₋₆ alkyl.5. The compound according to claim 4 wherein L is a bond or is


6. A pharmaceutical composition comprising the compound of claim 1 andone or more pharmaceutically acceptable excipients.
 7. A method forproducing an amino terminal polysaccharide derivative of GCSF accordingto claim 1, which method comprises covalently coupling an anionicpolysaccharide comprising 2-200 saccharide units attached to theamino-terminus of GCSF joined to one another through α2,8 or α2,9linkages.
 8. The method according to claim 7 wherein the anionicpolysaccharide has a reactive aldehyde group which reacts with the GCSFand the coupling reaction is carried out under reducing conditions. 9.The method according to claim 8 wherein the reactive aldehyde group isat the non-reducing end of the anionic polysaccharide.
 10. The methodaccording to claim 7 wherein the polysaccharide reacts with an aminegroup of the GCSF.
 11. The method according to claim 10 wherein thepolysaccharide reacts with a terminal amine group of the GCSF protein.12. The method accord to claim 10 wherein the amine is derived from alysine amino acid side chain of the GCSF protein.
 13. The methodaccording to claim 7 wherein the anionic polysaccharide reacts with aterminal amine group of the GCSF in a first aqueous solution of pH of4.0-6.0; and the resultant polysaccharide derivative is purified in asecond aqueous solution of pH of 6.5-8.5.
 14. A compound which is apolysaccharide derivative of granulocyte colony-stimulating factor(GCSF) wherein the polysaccharide is anionic and comprises between 2 and200 saccharide units.
 15. The compound according to claim 14 wherein thepolysaccharide is selected from polysialic acid, heparin, hyaluronicacid or chondroitin sulphate.
 16. The compound according to claim 14wherein the GCSF is derivatised by the polysaccharide at the reducingterminal unit of the polysaccharide.
 17. A compound of formula (I)

XB is B—XH wherein B is GCSF and XH is NH₂ and is an amine group of alysine amino acid side chain of GCSF; L is a bond or a linking group;GlyO is an anionic saccharide unit; wherein the linking group, ifpresent, is of the formula—Y—C(O)—R¹—C(O)—; wherein R¹ is a difunctional organic radical selectedfrom the group consisting of alkanediyl, arylene, alkarylene,heteroaryiene and alkylheteroarylene, any of which may substituted byand/or interrupted by carbonyl, ester, sulfide, ether, amide and/oramine linkages; and Y is NR² or NR²—NR² wherein R² is H or C₁₋₆ alkyl.18. The compound according to claim 17 wherein L is a bond or is


19. The pharmaceutical composition comprising the compound of claim 14and one or more pharmaceutically acceptable excipients.
 20. A method forproducing a polysaccharide derivative of GCSF according to claim 14,which method comprises covalently coupling an anionic polysaccharidecomprising 2-200 saccharide units attached to GCSF and joined to oneanother through u2,8 or u2,9 linkages.
 21. The method according to claim20 wherein the anionic polysaccharide has a reactive aldehyde groupwhich reacts with the GCSF and the coupling reaction is carried outunder reducing conditions.
 22. The method according to claim 21 whereinthe reactive aldehyde group is at the nonreducing end of the anionicpolysaccharide.
 23. The method according to claim 20 wherein thepolysaccharide reacts with an amine group derived from a lysine aminoacid side chain of the GCSF protein.